Recent advances in genetic engineering have provided the requisite tools to transform plants to contain foreign genes. It is now possible to genetically improve plants to have unique characteristics of agronomic importance. Certainly, one such advantageous trait is controlled plant cell lethality. Expression of a lethal gene in a cell can be used to specifically kill or prevent development of that targeted cell type from within an organism or from within a population of cells. A lethal gene has potential applications for hybrid seed production, cell ablation, and negative (counter) selection.
To be generally useful, lethal genes must be expressible behind a variety of promoters, be effective in a variety of cell types and be cell autonomous. Both dominant and conditional (inducible) lethal genes have been described in the literature. Dominant lethal genes include the expression of a protein which is directly toxic such as ricin which inhibits ribosomes (Landel et al., 1988), diphtheria toxin A (Dip-A gene) which ADP-ribosylates elongation factor 2 (Palmiter et al., 1987) and RNAses and DNAses which degrade critical genetic molecules (Mariani et al., 1990). Dominant lethal genes may also include antisense and ribozyme genes which target RNA molecules for critical cellular processes. The advantage of such dominant lethal genes is good efficacy and strict cell autonomous expression. The use of dominant lethal genes is severely restricted when expression of the lethal gene is critical for the survival or reproduction of the organism preventing the development of true-breeding progeny.
A conditional lethal phenotype involves the expression of a gene product which is not lethal to a cell in a normal environment but becomes lethal in a controlled environment. A conditionally lethal gene might encode a protein which converts an inactive or less active protoxin into a cytotoxic product or encode a biological molecule which prevents expression of a protein normally present to protect the cell from a toxin. The following discussion on conditional lethal genes will be in the context of a gene which converts a protoxin substrate into a cytotoxic product. The conditional lethal gene has several advantages over a dominant lethal gene. First, true breeding progeny can be obtained which express the conditional lethal gene without ill effects on the plants in the absence of the protoxin. Second, the timing and specificity for cell lethality can be controlled by both the expression of the gene and the application of the protoxin. Third, the efficacy of the conditional lethal gene can also be controlled by modifications to the protoxin if the activating enzyme encoded by this gene has a broad substrate specificity. Examples of conditional lethal genes used in plant studies include nitrate reductase which converts chlorate to toxic chlorite (Mueller and Grafe, 1978) and alcohol dehydrogenase which activates allyl alcohol (Schwartz and Osterman, 1976). However, these enzymes are normally expressed by wild-type cells limiting their use to cells which carry null mutations in the wild-type genes. Heterologous dominant conditional lethal genes have the greatest potential for controlled cell lethality. The genes are by definition, non-lethal in the absence of the controlled application of a heterologous protoxin and utilize protoxins which are not substrates for normal cellular enzymes. In plants, the only example of a heterologous conditional lethal gene is the iaaH gene encoding indoleacetamide hydrolase which can convert non-toxic levels of naphthalene acetamide into toxic levels of the auxin, naphthalene acetic acid (Klee et al., 1987). Since naphthalene acetamide is itself toxic at high levels, the conditional lethal phenotype is difficult to control. Viral thymidine kinase is an example of a heterologous dominant conditional lethal gene in mammalian cells. Unlike the cellular thymidine kinase, the viral thymidine kinase protein is able to activate pyrimidine analogs such as acycolvir and gancyclovia into toxic products (Elion, 1978 and Mansour et al., 1988). As described below, a novel conditional lethal gene is needed in plant biology which, has no affect on cellular metabolism in the absence of protoxin, utilizes a protoxin which is several orders of magnitude less toxic than the activated toxin, has a unique substrate specificity distinct from plant cellular enzymes and which utilizes a protoxin inexpensive to synthesize on a large scale.